Composition of scalable thyrointegrin antagonists with improved blood brain barrier penetration and retention into brain tumors

ABSTRACT

Chemical compounds/compositions, methods of synthesis, and methods of use. The compounds/compositions are directed toward thyrointegrin antagonists conjugated to a polymer. The compounds/compositions further comprise an additional substituent also conjugated to the polymer. The compounds/compositions demonstrate increased uptake across the blood brain barrier along with increased retention therein and retention within tumor. The compounds/compositions may also include improved synthesis scalability, improved purity, improved aqueous solubility, and a solid product or intermediate. The compounds/compositions may demonstrate improved antiangiogenic effect and improved efficacy against conditions, particularly cancers, requiring blood brain barrier permeability, for example, glioblastoma (GBM).

TECHNICAL FIELD

The present disclosure relates generally to improved thyroid hormonereceptor antagonists (referred to as “thyrointegrin antagonists”)compounds along with compositions comprising the same, methods of usingsuch compounds and compositions for treating conditions, and methods ofsynthesis. More specifically the present disclosure relates to compoundscomprising alpha-V-beta-3 (αvβ3) integrin-thyroid hormone receptorantagonists conjugated to a polymer, wherein the polymer is alsoconjugated to a further substituent or functional group. The disclosedcompounds, and compositions utilizing the compounds, have improved bloodbrain barrier penetration and retention, improved synthesis scalability,aqueous solubility, and/or solid products or intermediates. Thecompounds are also subject to readily scalable purification, for exampleby normal phase chromatography. Due to the increased penetration acrossthe blood brain barrier and retention into brain tumors, the disclosedcompositions and compounds are especially effective for treating certainconditions, for example Glioblastoma, Gliomas, Astrocytoma, CNSLymphoma, Medulloblastoma, Meningioma, Metastatic Brain Tumors,Pituitary Tumors, Primitive Neuroectodermal (PNET), and OtherBrain-Related Conditions.

BACKGROUND

Integrins are a super-family of cell surface adhesion receptors, whichcontrol the attachment of cells with the solid extracellularenvironment, both to the extracellular matrix (ECM), and to other cells.Adhesion is of fundamental importance to a cell; it provides anchorage,cues for migration, and signals for growth and differentiation.Integrins are directly involved in numerous normal and pathologicalconditions, and as such are primary targets for therapeuticintervention. Integrins are integral transmembrane proteins,heterodimers, whose binding specificity depends on which of the 14α-chains are combined with which of the 8 β-chains. The integrins areclassified in four overlapping subfamilies, containing the β1, β2, β3 orαv chains. A cell may express several different integrins from eachsubfamily. In the last several decades, it has been shown that integrinsare major receptors involved in cell adhesion, and so may be a suitabletarget for therapeutic intervention. Integrin αvβ3 regulates cell growthand survival, since ligation of this receptor can, under somecircumstances, induce apoptosis in tumor cells. Disruption of celladhesion with anti-αvβ3 antibodies, RGD peptides, and other integrinantagonists has been shown to slow tumor growth such as the cyclicpeptide Cilengitide that failed in Phase 3 Glioblastoma trial because ofits limited blood brain barrier permeability and brain tumor retention.

Applicant has previously disclosed compounds and compositions comprisingnon-cleavable polymer conjugated with αvβ3 integrin thyroid antagonistsas well as related methods, for example in U.S. patent application Ser.No. 15/616,637, now U.S. Pat. No. 10,201,616 and U.S. patent applicationSer. No. 16/223,176, the entire contents of both of which are herebyincorporated by reference.

Further, Applicant has previously disclosed compounds, compositions andmethods comprising αvβ3 integrin thyroid antagonists and targets of thenorepinephrine transporter or the catecholamine transporter (forexample, benzyl guanidine or derivatives) as well as related methods,for example in U.S. patent application Ser. No. 15/950,870, now U.S.Pat. No. 10,328,043 and U.S. patent application Ser. No. 16/398,342, theentire contents of both of which are hereby incorporated by reference.

While the compounds, compositions and methods described in theseprevious applications and issued patents were improvements to thethen-existing state of the art, such compounds and compositions maysuffer from one or more drawbacks including low blood brain barrierpermeability, poor synthesis scalability, lack of aqueous solubility,and the lack of formation of solid product or intermediate. Purificationmay also present difficulties. The disclosed compounds and compositionscomprising these compounds include improvements in these areas anddemonstrate unexpected efficacy in treating glioblastoma, other braintumors, and similar conditions.

Blood brain barrier permeability is important for targeting certainconditions, for example gliomas, meningiomas, pituitary adenomas,vestibular schwannomas, and medulloblastomas. Glioblastoma (glioblastomamultiforme or GBM) is one specific example of such a condition thatrequires blood brain barrier permeability for effective treatment. It isconventional in the art that drug delivery methods having improved bloodbrain barrier permeability would be beneficial. See e.g., Bhowmik A,Khan R, Ghosh M K. Blood Brain Barrier: A Challenge for EffectualTherapy of Brain Tumors. BioMed Research International, Volume 2015;Upadhyay R K. Drug Delivery Systems, CNS Protection, and the Blood BrainBarrier. BioMed Research International, Volume 2014. The improvedcompounds, compositions, and methods described herein demonstrateimproved blood brain barrier permeability and offer vastly improvedefficacy for these types of conditions. Further, the improved compounds,compositions, and methods described herein demonstrate improvedretention within the brain, particularly at the site of tumors locatedwithin the brain. This improved retention provides a further increasedefficacy in treating such conditions. The improved compounds,compositions, and methods described herein also demonstrate improvedscalability and solubility and may yield a solid product orintermediate.

A compound or composition such as those described herein comprising anαvβ3 integrin-thyroid hormone receptor antagonist (thyrointegrinantagonist) and having the described improved blood brain barrierpenetration and retention would be well received in the art, as wouldthe treatment methods using such compounds and/or compositions. Further,compounds or compositions having such improved blood brain barrierpenetration together with one or more of the described improvedsynthesis scalability, aqueous solubility, and/or formation of solidproducts or intermediates would likewise be well received in the art.

SUMMARY

According to one aspect, a compound comprises a thyrointegrinantagonist; a non-biodegradable polymer; a linker covalently bound tothe thyrointegrin antagonist and the non-biodegradable polymer via anon-cleavable covalent bond; and a substituent A bound to thenon-biodegradable polymer.

According to another aspect, a compound comprises a general formula:

wherein n1≥0; wherein n2 is 5-200; wherein R1-R4 and R9 areindependently selected from the group consisting of: H, Me, Et, iPr,nPr, nBu, iBu, secBu, tBu, C₅-C₁₂ n-alkyl, cyclopentyl, cyclohexyl,phenyl, F, Cl, Br, I, CN, CF₃, OCF₃, CHF₂, OCHF₂, SO₂Me, NO₂, —O-Alkyl,—O-Aryl, —CH₂—O-Alkyl, —CH₂—O-Aryl, Esters, and Amides; wherein R10-R13are each independently selected from the group consisting of hydrogen,iodine, and an alkane group; and wherein Y is selected from the groupconsisting of:

According to another aspect, a compound comprises a thyrointegrinantagonist conjugated to a polymer; and a substituted benzyl conjugatedto the polymer; wherein the compound is absorbed across the blood brainbarrier.

According to another aspect, a method of treating comprises providing acompound having a thyrointegrin antagonist and a substituted benzylconnected by a polymer; and administering a therapeutically effectiveamount of the compound to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some of the embodiments will be described in detail with reference madeto the following figures, in which like designations denote likemembers, wherein:

FIG. 1 depicts a general formula of an exemplary compound in accordancewith an embodiment of the invention;

FIG. 2 depicts a further detailed general formula of an exemplarycompound in accordance with an embodiment of the invention;

FIG. 3 depicts a further detailed general formula of an exemplarycompound in accordance with an embodiment of the invention;

FIG. 4 depicts exemplary Compound 2;

FIG. 5 depicts exemplary Compound 3;

FIG. 6 depicts exemplary Compound 4;

FIG. 7 depicts exemplary Compound 1;

FIG. 8 depicts exemplary Compound 5;

FIG. 9A depicts an embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 9B depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 9C depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 10A depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 10B depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 10C depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 11 depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 12A depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 12B depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 13A depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 13B depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 13C depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 13D depicts another embodiment of Substitution A from FIG. 1 inaccordance with an embodiment of the invention;

FIG. 14 depicts an exemplary synthetic pathway for Compound 1 inaccordance with an embodiment of the invention;

FIG. 15 depicts an exemplary synthetic pathway for exemplary Compound 2in accordance with an embodiment of the invention;

FIG. 16 depicts an exemplary synthetic pathway for exemplary Compound 3in accordance with an embodiment of the invention;

FIG. 17 depicts an exemplary synthetic pathway for exemplary Compound 4in accordance with an embodiment of the invention;

FIG. 18 depicts an exemplary synthetic pathway for exemplary Compound 5in accordance with an embodiment of the invention;

FIG. 19 depicts expression levels of integrin αvβ3 in glioblastomacancer cells analyzed by flow cytometry;

FIG. 20 depicts the results of a parallel artificial membranepermeability assay (PAMPA) for exemplary Compounds 1-4;

FIG. 21 depicts fluorescence intensity of exemplary Compound 5 indifferent organs and brain with and without glioblastoma;

FIG. 22 depicts levels of exemplary Compound 2 present in brain tissueover time following subcutaneous administration in mice;

FIG. 23 depicts plasma and brain levels of exemplary Compound 2 overtime following subcutaneous administration in cynomolgus monkeys at 15mg/Kg, s.c. daily for 14 days where brain tissues excised, and bloodsamples withdrawn for analysis by LC/MS/MS;

FIG. 24 depicts the antiangiogenic effect of exemplary Compound 2 in thepresence of various growth factors;

FIG. 25 depicts the antiangiogenic effect only against VEGF, and lackthereof against other growth factors, of known compound Avastin® in thepresence of various growth factors;

FIG. 26 depicts bioluminescent signals of GBM tumors in the brain;

FIG. 27 depicts blood brain barrier uptake of exemplary Compound 5 inthe brain in mice with and without GBM tumor;

FIG. 28A depicts blood brain barrier uptake of exemplary Compound 5 inthe brain in mice with and without GBM tumor, wherein exemplary Compound5 is administered along with compounds that are used in humans and maypotentially compete for uptake and retention;

FIG. 28B also depicts blood brain barrier uptake of exemplary Compound 5in the brain region with and without GBM tumor, wherein exemplaryCompound 5 is administered along with compounds that are used in humansthat may potentially compete for uptake and retention;

FIG. 29 depicts uptake of exemplary Compound 5 in brain in mice with andwithout tumor, as well as drug accumulation, or lack thereof, in otherorgans;

FIG. 30 depicts the effect of varying dosages of exemplary Compound 2 ontumor weight in mice with GBM xenografts;

FIG. 31 depicts the effect of varying dosages of exemplary Compound 2 ontumor cell luminescent signal intensity in mice with GBM xenografts;

FIG. 32 depicts the effect of a 6 mg/kg dose of exemplary Compound 2 ontumor weight compared with a 75 mg/kg dose of known compound Cilengitidein mice with GBM xenografts; and

FIG. 33 depicts the effect of a 6 mg/kg dose of exemplary Compound 2 ontumor cell luminescent signal intensity compared with a 75 mg/kg dose ofknown compound Cilengitide in mice with GBM xenografts.

DETAILED DESCRIPTION

A detailed description of the hereinafter-described embodiments of thedisclosed composition and method is presented herein by way ofexemplification and not limitation with reference to the Figures.Although certain embodiments are shown and described in detail, itshould be understood that various changes and modifications might bemade without departing from the scope of the appended claims. The scopeof the present disclosure will in no way be limited to the number ofconstituting components, the materials thereof, the shapes thereof,colors thereof, the relative arrangement thereof, etc., and aredisclosed simply as an example of embodiments of the present disclosure.A more complete understanding of the present embodiments and advantagesthereof may be acquired by referring to the following description takenin conjunction with the accompanying drawings, in which like referencenumbers indicate like features.

As a preface to the detailed description, it should be noted that, asused in this specification and the appended claims, the singular forms“a”, “an” and “the” include plural referents, unless the context clearlydictates otherwise.

Overview

Embodiments of the present disclosure describe new chemical compounds,compositions comprising the new chemical compounds, methods of synthesisthereof, and methods of treatment using such compounds and compositions.

The compounds disclosed herein (including but not limited to theexemplary compounds such as Compound 2, Compound 3, Compound 4, andCompound 5 described in detail below, along with compositions preparedfrom such compounds) demonstrate improved blood brain barrierpenetration and retention into brain tumors. Further, these compoundsand their respective compositions show an unexpected increase inefficacy against brain tumors and other conditions, for exampleglioblastoma (GBM).

The unexpected increase in efficacy against these conditions may be dueto a complex of factors—including active transport across the bloodbrain barrier, overexpression of integrin αvβ3 in GBM and similarconditions, and effect of further substituents on the thyrointegrinantagonist on uptake/retention.

First, the compounds and compositions described herein comprise athyrointegrin antagonist. Thyrointegrin antagonists such as thosedescribed herein may be subject to active transport across the bloodbrain barrier by thyroid binding proteins. A discussion of the transportof thyroid hormone and its analogs in the brain may be found at Wirth EK, Schwiezer U, Kohrle J. Transport of thyroid hormone in brain.Frontiers in Endocrinology. June 2014, Volume 5, Article 98. Drugdelivery methods having improved blood brain barrier permeability may bedifficult to achieve; however, Applicant's disclosed compounds andcompositions are actively transported across the blood brain barrier andthus reach the intended target site for therapeutic activity.

Second, the compounds and compositions described herein may be retainedwithin the blood brain barrier due to overexpression of integrin αvβ3 onGBM and similar conditions. For example, expression of αvβ3 in GBM mayreach levels of 80-97% as shown in FIG. 19. Like other thyrointegrinantagonists, the disclosed compounds may bind to this integrin bindingsite. Thus, in addition to being transported into the brain, thedescribed compounds and compositions bind to the tumor cells and may beretained within the blood brain barrier and retained at the intendedtarget site. Further, this reduces any unintended effect on non-tumortissue.

Third, as described in more details below, the compounds andcompositions described herein include additional functional groups.These additional functional groups may also be conjugated to thepolymer. For example, in embodiments having linear polymers, theadditional functional group may be conjugated on the opposite side ofthe polymer than the thyrointegrin antagonist. Non-linear polymers mayalso be used. The additional functional groups may further improve bloodbrain barrier penetration and retention and may also improve thescalability and/or solubility. For example, the increased uptake is notvia passive transport since (as shown by FIG. 20 below) analysis bypassive transport parallel artificial membrane permeability assay(PAMPA) shows low permeability of all derivatives in the absence ofthyroid binding proteins. Instead, the additional functional groupincrease active transport, for example, by making the thyrointegrinantagonist (transporter target) more accessible. PAMPA was carried outfor PMT36 and related compounds 2-4 where all exhibited low permeability(less than 1.5×E−6 cm/s). Similarly, low permeability of <1.5 E−6 cm/swas shown with Compound 5 as well as P-bi-TAT (a compound discussed inU.S. patent application Ser. No. 15/616,637, now U.S. Pat. No.10,201,616, and U.S. patent application Ser. No. 16/223,176). Theresults suggest that these compounds do not permeate through the bloodbrain barrier via passive diffusion, which clearly suggest that BBBpermeability to be facilitated mainly via the active transport systemusing thyroid binding proteins in blood such as transthyretin (TTR),which deliver the bound complex across the blood brain barrier.

Exemplary compounds will now be discussed in more detail along withadditional background information regarding potential thyrointegrinantagonists and polymers that may be used in embodiments of theinvention.

As discussed in U.S. patent application Ser. No. 15/616,637, now U.S.Pat. No. 10,201,616, and U.S. patent application Ser. No. 16/223,176,incorporated by reference above, compounds or compositions comprising anαvβ3 integrin-thyroid hormone receptor antagonist may include ananti-angiogenic thyroid hormone or derivative thereof conjugated via anon-cleavable linker to a polymer, forming a single chemical entitywhich may considered a micro molecule or macromolecule (depending on thesize of the polymer covalently bound to the thyroid hormone orderivative thereof). The size of the single chemical entity and thestrength of the non-cleavable covalent bond may be advantageous forpreventing the thyroid hormone or derivative thereof from entering cellscomprising a cell surface receptor of the integrin αvβ3 variety. Due tothe size of the attached polymer, and the inability of the surroundingenvironment of the cell to cleave the strong, uncleavable covalent bondsof the thyroid hormone from the polymer, the thyroid hormone portion ofthe described chemical entities may be unable to be internalized withinthe nucleus of the cells which the thyroid hormone or derivative thereofmay interact. Accordingly, the thyroid hormone portion may interact withthe cells non-genomically and avoid genomic interactions that may becaused by thyroid hormones or derivatives thereof entering a cell andinteracting with the nuclear receptors of the cellular nucleus.

As discussed in U.S. patent application Ser. No. 15/616,637, now U.S.Pat. No. 10,201,616, and U.S. patent application Ser. No. 16/223,176,incorporated by reference above, compounds or compositions comprising anαvβ3 integrin-thyroid hormone receptor antagonist may be synthesized toinclude, but are not limited to entities comprising non-biodegradablepolymers such as polyethylene glycol (PEG) (1,000-15,000 Daltons, forexample between 4,000-8,000 Daltons), α, β, or γcyclodextrins, chitosan,alginic acid or hyaluronic acid, conjugated via non-cleavable linkercomprising an amine or triazole bond, without short chain of PEG(100-800 M.W.) to an αvβ3 thyroid antagonist. Embodiments of the thyroidantagonists conjugated to the polymers may include tetraiodothyroaceticacid (tetrac), triiodothyroacetic acid (triac), derivatives thereof andvariations thereof. Examples of one or more variations of the thyroidhormone antagonists comprising tetrac and triac may include, in someembodiments Diaminotetrac (DAT) or Diamnotriac (DATri) (hereinafter maybe referred to interchangeably as “DAT”), Monoaminotetrac (MAT) orMonoaminotriac (MATri) (hereinafter referred to interchangeable as“MAT”), Triazoletetrac (TAT) or Triazoletriac (TATri) (hereinafterreferred to interchangeably as “TAT”), derivatives thereof or otherthyroid antagonist known by those skilled in the art.

As discussed in U.S. patent application Ser. No. 15/616,637, now U.S.Pat. No. 10,201,616, and U.S. patent application Ser. No. 16/223,176,incorporated by reference above, compounds or compositions comprising anαvβ3 integrin-thyroid hormone receptor antagonist have been furthersynthesized and characterized as DAT, MAT, or TAT conjugated todifferent molecular weights of Polyethylene Glycol (1,000 to 15,000Dalton). We have scaled up embodiments of the relatively most soluble,PEG-DAT (P-Mono-DAT, P-bi-DAT) and PEG-TAT (P-Mono-TAT, P-bi-TAT), forbiological characterization in various in vitro and in vivo biologicalsystems. Chemical labelling of DAT or TAT and PEG-DAT or PEG-TAT as wellas C-DAT and C-TAT for imaging and cellular kinetics. Data revealed thatpolymer conjugation to DAT or TAT resulted in the restriction of cellnuclear uptake of those polymers conjugated DAT or TAT versus intensecell nuclear uptake of DAT or TAT. The result of this unique cellulardistribution lead to the lack of genomic action of the polymerconjugated DAT, MAT or TAT versus the non-conjugated ones. OtherPolymers such as Hyaluronic, Alginic acid, Chitosan conjugated to DAT,MAT or TAT with or without short chain short chain PEG (100-1,000Dalton) are described. Additional Polymer conjugation to DAT, MAT or TATwere synthesized using bi-functional or tetra-function PEG may include,but it could also include other branched PEG up to 8 chains.

As discussed in U.S. patent application Ser. No. 15/616,637, now U.S.Pat. No. 10,201,616, and U.S. patent application Ser. No. 16/223,176,incorporated by reference above, compounds or compositions comprising anαvβ3 integrin-thyroid hormone receptor antagonist may have multipletypes of utility for treating a plurality of different diseasesmodulated by angiogenesis or the inhibition thereof. The compositions,in view of presence of the thyroid antagonist present in the describedcompositions, may each have an affinity for targeting the integrinreceptor αvβ3 located on numerous types of cells found throughout thehuman body and various animal bodies. For example, compositions may beuseful for treating angiogenesis-mediated disorders such as Cancer(Solid tumors and Liquid tumors) in humans or mammals. Cancers mayinclude Glioblastoma, pancreatic, ovarian, breast, prostate, bladder,lung and liver cancer. Liquid tumors may also include acute myeloidleukemia, multiple myeloma, Lymphoma and chronic lymphocytic leukemia.The compositions may further treat ocular disorders (DiabeticRetinopathy and Age-related Macular Degeneration), inflammatorydisorders (arthritis, osteoarthritis), atherosclerosis lesions, anddermatology (Rosacea, Psoriasis, skin cancer) which may each be mediatedor dependent upon the generation of new blood cells via angiogenesis topersist and the treatment thereof may be dependent antagonizing theformation of new blood vessel to slow or eliminate the angiogenicpathways.

The compounds and compositions disclosed herein improve upon Applicant'spreviously disclosed compounds and compositions in that they may achieveone or more of effective blood brain barrier penetration and retention,good synthesis scalability, good aqueous solubility, and yield a solidproduct amenable to scalable purification.

Reference may be made herein to specific thyrointegrin compounds, forexample, tetrac, triac, etc. These phrases include derivatives of suchcompounds in accordance with the full teachings of this disclosure, evenwhere such derivatives are not specifically listed.

Referring to the drawings, FIG. 1 depicts an embodiment of a generalformula 100 comprising a thyrointegrin antagonist 110 joined to asubstituent 120 (depicted generally as “A”), via a linker 130.Hereinafter, the substituent may be referred to as substituent A,substituent 120, or as substituent A 120. FIG. 1 depicts a carboxylicacid form of the general formula 100, as may other Figures present inthis application. As would be apparent to one skilled in the art, a salt(e.g. a sodium salt) of the general formula 100 may also be used.

In the depicted embodiment, the linker 130 comprises a spacer 132 and apolymer 131. The linker 130 resists biodegradation such that the linkerremains uncleaved under physiological conditions. In one embodiment, thespacer 132 comprises a CH₂ unit and an adjacent repeating linkage ofmethylene (CH₂) units which may be defined by n1 repeats wherein n1 isan integer that is ≥0. In other embodiments, n1 may be ≥1, ≥2 or ≥3. Thelinker 130 further comprises a moiety “Y.” Embodiments of the moiety“Y”, may in some instances be may be an amine. For example, the moiety Yof the general formula may be a divalent alkane having one amine groupor a divalent alkane having two amine groups as known from Applicant'sprevious applications. In another embodiment, the moiety Y may be atriazole as shown by the example of general formula 102 shown in FIG. 3.The polymer 131 may comprise a polyether such as polyethylene glycol(PEG). Other polymers may be used, including chitosan, alginic acid,hyaluronic acid, and other polymers. In embodiments using PEG as thepolymer 131, the polymer may have a molecular weight between 200 and4,000 g per mole.

The term thyrointegrin antagonist describes a compound that has theability to inhibit or antagonize one or more thyroid hormone receptorsknown by a person skilled in the art, for example the integrin family ofthyroid hormone receptors, such as the thyroid hormone cell surfacereceptor αvβ3. The thyrointegrin antagonist 110 may be ananti-angiogenic thyroid hormone or a thyroid hormone receptorantagonist. For example, the thyrointegrin antagonist 110 may be analpha-V-beta-3 (αvβ3) integrin-thyroid hormone receptor antagonist.

Specific embodiments of the thyrointegrin antagonist 110 may includetetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac),derivatives thereof and variations thereof. Examples of one or morevariations of the thyrointegrin antagonist comprising tetrac and triacmay include, in some embodiments Diaminotetrac (DAT) or Diaminotriac(DATri) (hereinafter may be referred to interchangeably as “DAT”),Monoaminotetrac (MAT) or Monoaminotriac (MATri) (hereinafter referred tointerchangeable as “MAT”), Triazoletetrac (TAT) or Triazoletriac (TATri)(hereinafter referred to interchangeable as “TAT”), derivatives thereofor other thyroid antagonist known by those skilled in the art.Thyrointegrin antagonists may be of the types described in U.S. patentapplication Ser. No. 15/616,637 now U.S. Pat. No. 10,201,616 and U.S.patent application Ser. No. 16/223,176, the entire contents of both ofwhich are hereby incorporated by reference and/or in U.S. patentapplication Ser. No. 15/950,870 now U.S. Pat. No. 10,328,043 and U.S.patent application Ser. No. 16/398,342, the entire contents of both ofwhich have been incorporated by reference. As described in thosedocuments, in some embodiments of the thyrointegrin antagonist 110, thevariables depicted as R10, R11, R12, and R13 may each independently besubstituted for molecules such as hydrogen, iodine, and alkanes. In someembodiments, the alkanes have four or fewer carbons.

In embodiments of the invention, the substituent A 120 may be orcomprise an aryl group and/or an aromatic group. For example, inembodiments, the substituent A 120 may comprise a benzyl group, a phenylgroup, and the like. In some embodiments the substituent A 120 maycomprise a substituted benzyl group. In further embodiments, aheterobenzyl group may be used. Still further, 5 membered ringheteroaryls, fused heteroaryls, qinolines, and indoles may also be used.The heteroaryls may comprise heteroarylmethyls. In further embodimentsthe substituent A 120 may comprise esters and amides.

FIG. 2 depicts a general formula 101 in which the substituent A 120 isdepicted as comprising an aromatic ring according to embodiments. Thesubstituent A 120 comprising the aromatic ring may be, for example, asubstituted benzyl group. In embodiments, the substituent A 120comprising the aromatic ring may be substituted at one or more of R1,R2, R3, R4, and R9. In some embodiments of the substituent A 120comprising the aromatic ring, the variables depicted as R1, R2, R3, R4,and R9 may be each independently be substituted for molecules such ashydrogen, iodine, fluorine, bromine, a methoxy group, a nitro group, anamine group, and a nitrile group. For example, in some embodiments ofthe substituent A 120 comprising the aromatic ring, the variablesdepicted as R1, R2, R3, R4, and R9 may be each independently besubstituted for molecules of hydrogen, iodine, fluorine, bromine, amethoxy group, a nitro group, an amine group, and a nitrile group asdescribed in Table 2 of U.S. patent application Ser. No. 15/950,870 nowU.S. Pat. No. 10,328,043 and U.S. patent application Ser. No.16/398,342. Still further, the variables depicted as R1, R2, R3, R4, andR9 may be substituted with alkyls, aryls, halos, amides, and the like.

As shown in FIG. 3, in some embodiments including the depicted generalformula 102, the variables R1, R2, R3, R4 may be substituted formolecules of hydrogen while R9 may be substituted for a differentmolecule or group, “Z” in the depicted embodiment. Thus, the substituentA 120 may comprise an aromatic ring as discussed above, and may morespecifically comprise a substituted benzyl group 122 in which themolecule or group Z has been substituted for hydrogen at R9.Alternatively, as shown in FIG. 6, in some embodiments, variables R1 andR2 or other variables may be substituted instead of R9.

Each of the exemplary Compounds shown in FIGS. 3-8 (including exemplaryCompounds 1-5) comprises tetrac as the pertinent thyrointegrin αvβ3receptor antagonist, polyethylene glycol as the pertinent linker, and atriazole as the included Y moiety.

Turning more specifically to exemplary Compounds 2-5, these may bebroadly referred to as X-PTAT, wherein the substituent A 120 is nowspecified as a substituted benzyl group (such as the substituted benzylgroup 122 shown in FIG. 3, fluorobenzyl or chlorobenzyl as shown inFIGS. 4 and 5, respectively, or a different substituted benzyl groupsuch as that shown in FIG. 6) and referred to as X, P is the polymer orpolyethylene glycol, and TAT refers to triazole tetrac. Further, theymay be referred to as X-PMTAT, wherein the substituent A 120 is nowspecified as the substituted benzyl group and referred to as X, P is thepolymer or polyethylene glycol, and MTAT refers to monotriazole tetrac.Still further, they may be referred to as X-PMT, wherein the substituentA is now specified as the substituted benzyl group and referred to as X,P is the polymer or polyethylene glycol, and MT again refers tomonotriazole tetrac.

These designations are in contrast to the compound depicted in FIG. 7,namely Compound 1. This Compound 1 comprises a specific embodiment of athyrointegrin antagonist conjugated to a polymer such as is described inU.S. patent application Ser. No. 15/616,637 now U.S. Pat. No. 10,201,616and U.S. patent application Ser. No. 16/223,176. This compound may bereferred to as Compound 1 and also as PTAT, PMT, or PMTAT. As discussedabove, these designations indicate the inclusion of polymer conjugatedto (mono)triazole tetrac, in this case conjugated only to a methylgroup, and without conjugation to a functional group such as substituentA 120 described above (or the more specific examples such as thesubstituted benzyl group).

Turning back to embodiments of the currently disclosed invention, suchas those shown in FIGS. 4 and 5, the variable R9 may be substituted fora halogen. For example, R9 may be substituted for a fluorine molecule asshown in FIG. 4. This structure is referred to as Compound 2 and mayalso be referred to as fluorobenzyl conjugated to triazole tetrac bypolyethylene glycol or alternatively as fb-PTAT, fb-PMTAT, or fb-PMT. Inanother embodiment, R9 may be substituted for a chlorine molecule asshown in FIG. 5. This structure is referred to as Compound 3 and mayalso be referred to as chlorobenzyl conjugated to triazole tetrac bypolyethylene glycol or alternatively as cb-PTAT, cb-PMTAT, or cb-PMT.

In still further embodiments such as that shown in FIG. 6, the variablesR1 and R2 may be substituted. For example, R1 and R2 may be substitutedfor tert-butyl groups. This structure is referred to as Compound 4 andmay also be referred to as di-tbutylbenzyl conjugated to triazole tetracby polyethylene glycol or alternatively as Dtbb-PTAT, Dtbb-PMTAT, orDtbb-PMT.

Additional embodiments may include a dye, marker, label, or the like,for example, for imaging purposes. The dye, marker, or label may be onthe substituted benzyl in some embodiments. For example, FIG. 8 depictsa labeled polymer conjugated monotetrac (PMT) derivative referred to asCompound 5. In this example, substituent A 120 comprises a dye marker,for example BODIPY. Compound 5 may also be referred to as BODIPY-PMT.

As discussed above, additional embodiments of the substituent A 120 arealso contemplated. For example, as shown in FIGS. 9A, 9B, and 9C,additional ring structures are disclosed for substituent A 120. In theseexamples, R8 may be equal to H, Me, Et, and the like. R1-R7 may beindependently selected from H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu,C₅-C₁₂ n-alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br, I, CN, CF₃,OCF₃, CHF₂, OCHF₂, SO₂Me, NO₂, —O-Alkyl, —O-Aryl, —CH₂—O-Alkyl,—CH₂—O-Aryl, esters, amides, and the like. Ester substitution may beselected from the following:

and amide substitution may be selected from the following:

wherein R9 and R10 are independently selected from H, Alkyl, Aryl, andthe like.

Still further, as discussed above, additional embodiments of thesubstituent A 120 may include heterobenzyls such as those shown in FIGS.10A through 10C. Moreover, 5-membered ring heteroaryls, fusedheteroaryls, quinolines, indoles, and the like may also be used. R1-R5and R8 may be substituted as described above.

Phenoxy groups such as that shown in FIG. 11 may also be used asembodiments of substituent A 120. Again, R1-R5 may be substituted asdescribed above.

In additional embodiments, substituent A 120 may comprise an amide suchas those depicted in FIGS. 12A and 12B. R9 and R10 may be substituted.

Esters may also be used as substituent A 120 in embodiments. Forexample, esters such as those depicted in FIGS. 13A-13D may be used.

As described above, in embodiments varying types of polymer may be used,as well as various molecular weights of polymer. In embodiments,monodisperse polymers may be used to increase ease of analysis andscalability relative to polydisperse polymers. Further, as shown inexemplary Compounds 2-4, embodiments may include a relatively largepolymer such as PEG36. Large monodisperse polymers such as monodispersePEG36 may contribute to solubility and analysis of the exemplarycompounds. Further, such large monodisperse polymers may increasescalability by yielding a relatively large solid product amenable topurification. Thus, embodiments comprising PEG36 conjugated to monotriazole tetrac may have simplified synthesis and scalability whencompared with other embodiments. In some embodiments, the polymer mayhave a molecular weight of approximately 4,000 Daltons, for example,4,000±10%. Still further, these large monodisperse polymers maycontribute to the increased active transport of the compound, forexample, by making the thyrointegrin antagonist (transporter target)more accessible.

Synthesis of the specific exemplary compounds described herein(Compounds 1-5) is demonstrated below. The synthesis description isprovided only as examples and is not intended to limit the disclosure.These example uses propargylated tetrac (PGT). Preparation of PGT or aderivative thereof from tetrac is described in U.S. patent applicationSer. No. 15/616,637 now U.S. Pat. No. 10,201,616.

Example 1: Synthesis of Compound 1 (PMT)

Compound 1 and similar compounds/compositions were described in U.S.patent application Ser. No. 15/616,637 now U.S. Pat. No. 10,201,616(see, for example, FIGS. 7c and 8 (compound 730)), and may be preparedas described therein. However, Applicant also provides the followingsample method:

FIG. 14 depicts an overview of a synthetic pathway for Compound 1. Theindividual steps of the scheme of synthesis of Compound 1 will bedescribed in more detail below.

Step 1: 1 g (0.625 mmol) of monodisperse MeoPEG₃₆OH (PurePEG, San DiegoCalif.) and 0.285 ml (4 eq.) TEA were dissolved in 10 ml DCM. 238 mg (2eq.) of TosCl was added portionwise over 10 min with stirring, andstirred overnight. 10 mL of DCM was added, and the mixture washed 2×with 5 mL of saturated ammonium chloride, 2× with 5 mL of saturatedsodium bicarbonate, 1× with 5 mL of saturated brine, and the solvent wasstripped under vacuum. The solid was dissolved in 10 mL hot THF add anequally volume of hot hexane was added. The liquid was decanted from asmall amount of insoluble material and the product was allowed toprecipitate at −20 C overnight. The product was filtered, washed withhexane, and dried under vacuum yielding 995 mg (90%) of product.

Step 2: 990 mg MeOPEG₃₆OTs (0.565 mM) was dissolved in 5 ml CH₃CN. 110mg (3 eq) of sodium azide was added, and the mixture was heated at 70 Covernight with stirring. The reaction was then cooled and most of theacetonitrile was removed under reduced pressure, and the residue waspartitioned between 10 mL each of DCM and water. The aqueous layer wasextracted 3× with 5 mL portions of DCM, and the combined organic layerswere washed with 5 ml each of water and saturated brine. The solvent wasstripped under reduced pressure, and the material was precipitated fromTHF/hexane in a procedure similar to the previous step yielding 832 mg(89%) of product.

Step 3: 830 mg (505 mmol) MeO-PEG₃₆-N₃, 474 mg(4-{3,5-Diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)aceticacid (1.2 eq), and 13.7 mg TBTA (5%) were dissolved in 20 ml THF.Dissolve 12.6 mg (0.1 eq) copper sulfate hydrate and 60 mg (0.6 eq)sodium ascorbate in 5 ml of water and add to the THF solution. Stirunder N₂ for 16 h, then decant the liquid from the small amount of bluesolids on the bottom of the flask. Strip the THF from the solution underreduced pressure, add 10 ml of water, acidify to pH 3 with dilute HCl,and extract 3×40 mL with DCM. Wash the combined organic layers 3× with 5mL of saturated EDTA solution, then 5 mL of saturated brine. Strip theDCM under reduced pressure, and dissolve the remainder in 20 ml of warmTHF. Add 20 ml of hot hexane, and warm until almost everything isdissolved. Cool to room temperature, then decant the liquid from thesmall amount of solid and oil on the bottom of the vial. Allow themixture to precipitate at −20 C overnight, filter off the solids, andwash with cold hexane. Dry the remaining white solid under reducedpressure, 685 mg. Dissolve in 6.5 mL H2O with 2.5 mL of 1M NaOH and 1 mLof saturated sodium chloride. Wash 2×25 mL with 3:2 hexane:DCM. Add 1 mlmore of saturated NaCl and wash with another 2×25 mL of 3:2 Hexane:DCM.The aqueous layer was acidified to pH 2.0 with dilute HCl and extracted3×25 mL with DCM. The combined organic layers were washed with saturatedsodium chloride, and the solvent was removed under reduced pressure. Theresidue was precipitated from 20 mL of THF and 20 mL of hexane, yielding491 mg of product. ¹H NMR (600 MHz, DMSO D₆) d (PPM): 8.258 (s, 1H),7.850 (s, 2H), 7.195 (s, 2H), 5.015 (s, 2H), 4.581 (br. s, 2H), 3.851(br. s, 2H), 3.6-3.3 (m, 144H), 3.239 (S, 3H). MS m/z 2452.2 (M+Na),1215.4 (M+2H), 810.2 (M+3H), 608.2 (M+4H).

Example 2: Synthesis of Compound 2 (fb-PMT)

FIG. 15 depicts an overview of a synthetic pathway for Compound 2. Theindividual steps of the scheme of synthesis of Compound 2 will bedescribed in more detail below.

Step 1: 250 mg HO-PEG36-azide (0.155 mmol, PurePEG, San Diego Calif.)was added to 19 mg 60% NaH (3 eq) in 5 ml of THF. The mixture wasstirred for 30 min, then 58 uL of 4-(fluorobenzyl)bromide (Aldrich) (3eq.) in 2 mL of THF was added dropwise. The mixture was stirred 18 h,then 2 mL of saturated sodium bicarb was added. The THF was stripped offunder vacuum, 10 mL of saturated brine was added, the mixture wasextracted 3× with 15 ml portions of DCM. The combined organic layerswere washed with 5 ml of saturated brine and the solvent was strippedoff under vacuum. The material was then chromatographed on 24 g ofsilica with 0-10% MeOH in DCM yielding 170 mg of material, >99% pure byHPLC.

Step 2: 170 mg (0.147 mmol) of 4-fluorobenzylPEGazide, 138 mg (0.176mmol)(4-{3,5-Diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)aceticacid, and 3 mg of TBTA were dissolved in 8 mL of THF. Add 3 mg CuSO4hydrate and 23 mg Na ascorbate in 2 mL water and stir 4 h under N2.Strip off the THF under vacuum, then add 5 ml saturated brine and 0.5 mlof 1M HCl. Extract 3×10 mL with DCM, wash 3×5 mL with saturated EDTA,once with 5 mL of saturated brine, and strip the solvent under vacuum.Dissolve the residue in 10 mL of warm THF, then add hexane until it juststarts to turn cloudy. Allow the product to precipitate overnight at −20C. Filter off the solid to get 180 mg of product. ¹H NMR (600 MHz, DMSOD₆) d (PPM): 8.246 (s, 1H), 7.879 (s, 2H), 7.361 (dd, 2H), 7.167 (m,4H), 5.013 (s, 2H), 4.575 (m, 2H), 4.466 (s, 2H), 3.847 (m, 2H), 3.640(s, 2H), 3.55-3.4 (m, 144H). MS m/z 1262.8 (M+2H), 842.5 (M+3H), 632.2(M+4H). The product can be further purified by chromatography on normalphase silica gel.

Example 3: Synthesis of Compound 3 (cb-PMT)

FIG. 16 depicts an overview of a synthetic pathway for Compound 3. Theindividual steps of the scheme of synthesis of Compound 3 will bedescribed in more detail below.

Step 1: 250 mg PEGazide (0.155 mmol) added to 19 mg 60% NaH (3 eq) in 5ml THF. Stir 30 min, then add 95.5 mg 4-chlorobenzylbromide (Aldrich) (3eq.) in THF dropwise. Stir 18 h, add saturated sodium bicarbonatesolution, strip off the THF under vacuum, add 10 mL sat brine, extract3× with 15 ml portions of DCM, wash the combined organic layers withsaturated brine, and strip off the solvent under vacuum. Chromatographwith 0-10% MeOH in DCM on silica gel. 190 mg.

Step 2: Dissolve 190 mg (0.111 mmol) chlorobenzylPEGazide, 131 mg(4-{3,5-Diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)aceticacid (1.5 eq), and 3 mg TBTA in 8 mL THF. Add 3 mg CuSO4 hydrate and 23mg Na ascorbate in 2 mL water, and stir 4 h. Strip off the THF undervacuum, then add 5 of saturated brine and 0.5 ml of 1M HCl. Extract 3×with DCM, wash 3× with saturated EDTA, once with brine, and strip thesolvent under vacuum. Dissolve in 10 mL of warm THF, then add hexaneuntil it just starts to turn cloudy. Precipitate the material in −20freezer, 180 mg product. ¹H NMR (800 MHz, D₂O) d (PPM): 8.369 (s, 0.3H),8,147 (s, 0.7H) 7.758 (s, 2H), 7.261 (m, 6H), 4.993 (s, 2H), 4.541 (m,2H), 4.451 (s, 2H), 3.849 (m, 2H), 3.640 (s, 2H), 3.65-3.54 (m, 142H),3.346 (s, 2H). MS m/z 1281.2 (M+2H), 854.9 (M+3H), 641.4 (M+4H). Theproduct can be further purified by chromatography on normal phase silicagel.

Example 4: Synthesis of Compound 4 (Dtbb-PMT)

FIG. 17 depicts an overview of a synthetic pathway for Compound 4. Theindividual steps of the scheme of synthesis of Compound 4 will bedescribed in more detail below.

Step 1: 250 mg HO-PEG36azide (0.155 mmol) was added to 19 mg 60% NaH (3eq) in 5 ml THF. Stir 30 min, then add 131 mg bromide (Aldrich) (3 eq.)in THF dropwise. Stir 18 h, add saturated sodium bicarbonate, strip offthe THF under vacuum, add 10 mL sat brine, extract 3× with 15 mlportions of DCM, wash the combined organic layers with saturated brine,and strip off the solvent under vacuum. Chromatograph with 0-20% MeOH inDCM on silica gel. 270 mg.

Step 2: 270 mg (0.147 mmol) of di-tbutylbenzylPEGazide, 171 mg(4-{3,5-Diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)aceticacid, and 4 mg TBTA were dissolved in 8 mL THF. Add 4 mg CuSO4 hydrateand 34 mg Na ascorbate in 2 mL water, and stir 4 h. Strip off the THFunder vacuum, then add 5 ml saturated brine and 0.5 ml of 1M HCl.Extract 3× with DCM, wash 3× with saturated EDTA, once with brine, andstrip the solvent under vacuum. Chromatograph with 0-10% MeOH in DCM onsilica gel. 310 mg. ¹H NMR (600 MHz, DMSO D₆, d (PPM): 8.248 (s, 1H),7.842 (s, 2H), 7.300 (s, 1H), 7.188 (s, 2H), 7.130 (s, 2H), 5.007 (s,2H), 4.564 (m, 2H), 4.455 (s, 2H), 3.843 (m, 2H), 3.6-3.3 (m, 144H),1.274 (s, 18H). MS m/z 1309.3 (M+2H), 873.4 (M+3H), 655.8 (M+4H).

Example 5: Synthesis of Compound 5 (BODIPY-PMT)

FIG. 18 depicts an overview of a synthetic pathway for Compound 5. Theindividual steps of the scheme of synthesis of Compound 5 will bedescribed in more detail below.

Step 1: 250 mg of NH2-PEG36-N3 were treated with 1.5 equivalents of BOCanhydride and 3 eq of triethyl amine in 5 mL of DCM. After stirring for18 h at room temperature the mixture was diluted with 20 ml of DCM andwashed with 0.1M HCl followed by saturated sodium bicarbonate andsaturated brine. The solvent was removed under reduced pressure, theresidue was dissolved in 5 mL of warm THF, and hexane was added untilthe mixture started to turn cloudy. The mixture was allowed to standovernight before filtering and washing with hexane. 240 mg of productwas recovered.

Step 2: 235 mg of the step 1 product, 171 mg(4-{3,5-Diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)aceticacid, and 4 mg TBTA were dissolved in 8 mL THF. Add 4 mg CuSO4 hydrateand 34 mg Na ascorbate in 2 mL water, and stir 18 h. Strip off the THFunder vacuum, then add 5 ml saturated brine and 0.5 ml of 1M HCl.Extract 3× with DCM, wash 3× with saturated EDTA, once with brine, andstrip the solvent under vacuum. The residue was dissolved in 5 mL ofwarm THF, and hexane was added until the mixture started to turn cloudy.The mixture was allowed to stand overnight before filtering and washingwith hexane. 220 mg of product was recovered.

Step 3: 215 mg of step 2 product was dissolved in 2 mL of DCM, and 2 mLof 5M HCl in dioxane was added. The mixture was stirred for 18 h, thesolvent was removed under reduced pressure, and the product was used asis for the next step.

Step 4: 18 mg of product from the last step (0.0076 mmol) was dissolvedin 1 ml of DCM with 10 uL of triethyl amine. 5 mg of BODIPY 630/650 NHSester (Thermo Fisher) dissolved in 100 ul of DCM was added. The mixturewas shaken for 18 hours, the solvent was removed under reduced pressure,and the residue was chromatographed with on silica gel with 0-20%methanol in DCM. 4.5 mg were recovered. ¹H NMR (800 MHz, CDCl3) d (PPM):8.221 (s, 1H), 8.058 (s, 1H), 7.850 (s, 2H), 7.642 (m, 3H), 7.513 (d,1H), 7,237 (m, 3H) 7.075 (m, 1H), 7.021 (m, 1H), 6.993 (m, 3H), 6.828(m, 1H), 6.729 (m, 1H), 5.218 (s, 2H), 4.619 (m, 2H), 4.570 (s, 2H),3.836 (m, 2H), 3.75-3.6 (m, 140H), 3.545 (m, 2H), 3.416 (m, 2H), 3.310(m, 2H), 2,223 (m, 2H), 1.688 (m, 2H), 1.603 (m, 2H), 1.359 (m, 2H). Theproduct had a retention time of 34.05 minutes on HPLC system 1: PursuitXRs 3 C18 column, Mobile phase A (water with 0.1% formic acid and 5%acetonitrile) and methanol (B). Flow rate was 1.0 mL/min, gradient waslinear from 50% B at 0 min to 95% B at 40-45 min, column temperature 25C.

Again, other synthetic pathways in addition to those described above maybe used to produce the exemplary compounds. Further, additionalcompounds may be generated using the techniques described above,modified as needed for the respective substitutions.

Methods of Use/Treating

As discussed above, the compounds and compositions described herein haveincreased uptake across the blood brain barrier and into the brain.Table 1 below demonstrates this increased uptake by showing averagebrain concentration for each of the exemplary Compounds 1-4.Concentrations are shown as recorded 3 h following administration.

TABLE 1 Brain penetration data Average Brain Concen- tration (ng/g),Compound Chemical Structure 3 hour m-PMT (1)

55.1 fb-PMT (2)

228 cb-PMT (3)

266 Dtbb-PMT (4)

922 BG-P-TAT

0.00

The data in Table 1 was generated using the following study:Compositions 1-4 were administered to C57BL/6 mice subcutaneously at 10mg/Kg. Each group contained 4 mice. The mice were then sacrificed 3hours post dosing and brain tissues were excised for bio analyticalmeasurement of Compounds 1-4 in the brain. Average concentrations areshown above and each of Compounds 2-4 demonstrated increased uptakecompared with Compound 1.

Further, each of Compounds 2-4 also demonstrate increased uptakecompared with BG-P-TAT. BG-P-TAT refers to benzyl guanidine conjugatedto tetrac via polymer PEG and was described, along with other compoundsand compositions comprising αvβ3 integrin thyroid antagonists andtargets of the norepinephrine transporter or the catecholaminetransporter, in U.S. patent application Ser. No. 15/950,870 now U.S.Pat. No. 10,328,043 and U.S. patent application Ser. No. 16/398,342. Thetested embodiment of BG-P-TAT used PEG36. As can be seen, BG-P-TAT doesnot penetrate the blood brain barrier and there is no detectable levelin the brain 3 h after administration. Again, this is in sharp contrastto the currently-disclosed compounds such as exemplary Compounds 2-4which demonstrate high levels of concentration in the brain.

Increased uptake of the presently disclosed compounds was determined tobe due to active transport rather than to passive permeability. Forexample, as shown in FIG. 20, analysis by passive transport parallelartificial membrane permeability assay (PAMPA) showed low permeabilityof each of Compounds 1-4. Thus, very little blood brain barrierpermeability is achieved without the presence of thyroid bindingproteins. Because passive permeability is unaffected and each ofCompounds 1-4 have the same transporter recognition element (thethyrointegrin antagonist, triazole tetrac), it would be expected thateach of these compounds would be a substrate for thyroid hormonetransporters and would likewise have the same or similar uptake into thebrain.

However, as demonstrated in Table 1 above, exemplary Compounds 2-4 showa marked and unexpected increase in brain concentration levels over bothCompound 1 and BG-P-TAT. Further, as is discussed in more detail below,this unexpected increase in brain uptake and concentration results insimilarly unexpected enhanced efficacy in treating conditions requiringblood brain permeability, including, for example, glioblastoma.

Further, as shown in FIG. 21, uptake increases substantially when tumoris present in the brain. As discussed above, this is due, at least inpart, to the binding of the compound/composition to the high expressionof αvβ3 by GBM tumors. Further, uptake is predominantly concentrated inthe brain to the exclusion of other organs as shown. In the data shownin FIG. 21, uptake was measured using Compound 5 (BODIPY-PMT).

FIG. 22 also depicts this initial uptake into the brain. As shown,exemplary Compound 2 (fb-PMT) demonstrates uptake and retention over a24 hour period. Again, a single subcutaneous injection was used in mice.The administered dose was 10 mg/Kg. The brain tissue in this exampledoes not include tumor cells.

Plasma concentrations and brain levels for exemplary Compound 2 (fb-PMT)in cynomolgus monkeys are depicted in FIG. 23. As can be seen, plasmaconcentration peaks between 1 and 4 hours. Brain levels are alsoincluded and Compound 2 is present at 72.3 ng/g and 80.5 ng/g (male andfemale cynomolgus monkey, respectively) 24 hours after the final dose ofa 14 day treatment regimen (15 mg/Kg, SC QD for 14 days) as measuredusing validated LC/MS/MS method.

In addition to good initial uptake, the disclosed compounds also havegood anti-angiogenic effect. For example, Exemplary Compound 2 (fb-PMT)demonstrates broad spectrum anti-angiogenic affect against differentgrowth factors as shown in FIG. 24. Specifically, Compound 2 iseffective at reducing the percentage of angiogenesis present in CAMModels, when administered in the presence of the following growthfactors: bFGF, VEGF, VEGF+bFGF, and bFGF+VEGF+HGF. Each of these growthfactors produces a 250% or greater increase in angiogenesis in the CAMModel; however, administration of Compound 2 (fb-PMT) at 1.0 μgdrastically reduces this increase to only slightly above baseline.

This broad spectrum anti-angiogenic effect is in contrast to existingtherapeutic compounds/compositions such as AVASTIN® (bevacizumab). Asshown in FIG. 25, AVASTIN® is primarily effective against VEGF alone,but does not meaningfully reduce the percent of angiogenesis for bFGF orHGF. Further, AVASTIN® does not demonstrate good effect when bFGF orbFGF and HGF are present along with VEGF. Referring back to FIG. 24,exemplary Compound 2 (fb-PMT) demonstrates inhibition of angiogenesisagainst all three growth factors, both alone and in combination.

Referring again to FIG. 21, exemplary Compound 5 (BODIPY-PMT) showssubstantially increased uptake when tumor (GBM) is present in the brain.Further, uptake is predominantly concentrated in the brain to theexclusion of other organs as shown. The study protocol will now befurther described.

Athymic female mice were used, with and without GBM in the brain. Micewith GBM received brain orthotopic implantation of U87-luc cells (1million cells). Compound 5 (BODIPY-PMT) (far-red fluorescence dye) wasinjected subcutaneously at 3 mg/Kg, s.c. Fluorescence signals weredetected (Ex/Em 630 nm/650 nm). The following table shows the fulltreatment groups:

TABLE 2 Fluorescence Protocol Mice (No Mice (U87- Treatment Tumor) luctumor) Control 4 4 Compound 5 (BODIPY-PMT) (3 mg/kg, s.c.) 4 4 L-T4* (20μg/kg, s.c.) 4 4 L-T4 (20 μg/kg) + Compound 5 (3 mg/kg, s.c.) 4 4Phenytoin* (1 mg/kg, s.c.) 4 4 Phenytoin (1 mg/kg, s.c.) + Compound 5 44 (3 mg/kg, s.c.) *L-T4 (thyroxine for hypothyroidism) and Phenytoin(antiseizure) bind to thyroid binding proteins

Fluorescence signals of Compound 5 were imaged after 1 h, 2 h, 6 h, and24 h. After termination, ex vivo fluorescence signals were imaged in thebrain and organs. Following the post-termination imaging, luciferasesubstrate was added to detect tumor luminescent signals in the brain.

FIG. 26 shows the bioluminescent signal of GBM (U870-luc) tumor in thebrain for the different treatment groups.

FIG. 27 shows the presence of Compound 5 (administered at 3 mg/Kg, s.c.)in the brain at the 1 h, 2 h, 6 h, and 24 h intervals. As shown,Compound 5 undergoes uptake into the blood brain barrier and is retainedat all intervals. Further, as shown Compound 5 is present atsubstantially higher levels in the animals with GBM tumors, furtherevidencing uptake across the blood brain barrier and retention into thetumor site.

FIG. 28A shows the blood brain barrier uptake and retention of exemplaryCompound 5 (3 mg/Kg) after 24 h. Again, Compound 5 is present atsubstantially higher levels in the animals with GBM, further evidencinguptake across the blood brain barrier and retention at the tumor site.Compound 5 is present and retained when administered alone and also whenadministered with other drugs crossing the blood brain barrier, such asthyroid hormone L-T4 and Phenytoin. Thus, the disclosedcompounds/compositions demonstrate good uptake and retention within thebrain even in the presence of drugs that may compete for uptake/binding.

FIG. 28B also shows fluorescence signal intensity of Compound 5 in thebrain region with and without GBM tumor. Again, Compound 5 wasadministered alone and in the presence of L-T4 and Phenytoin. As can beseen, uptake is substantially increased when tumor is present. Further,the increase in uptake is not diminished by the presence of L-T4 norPhenytoin. Thus, the disclosed compounds demonstrate good uptake intothe brain as well as high affinity binding and retention at the tumorsite.

FIG. 29 shows the brain uptake of a single dose (3 mg/Kg, SC) ofexemplary Compound 5 (fb-PMT) in mice with and without tumor. As can beseen, animals with implanted tumors demonstrate substantially increasedlevels of uptake into the brain. Further, FIG. 29 also showsaccumulation levels within other organs, including heart and lungs,liver, and kidneys. Drug accumulation is present in the liver in bothtest groups; however, animals with tumor do not demonstrate accumulationin the kidneys while animals without tumor do demonstrate accumulationin the kidneys. This is further evidence for retention at the tumorsite. FIG. 29 also shows luminescent signals of the GBM in the animalswith tumor compared to no signal in animals without tumor.

The foregoing FIGS. 21-29 demonstrate the increased blood brain barrieruptake and retention of the exemplary compounds. Thus, the disclosedcompounds and compositions comprising these compounds may be deliveredacross the blood brain barrier and specifically to tumor sites locatedwithin the brain. Further, the compounds may be used to target suchtumors while minimizing effect on healthy tissue.

The efficacy of these compounds/compositions with respect to GBM tumorswill now be described with reference to FIGS. 30-33. The study protocolis as follows: Nude mice having U87-luc xenografts were treated withvarying dosages of Compound 2 (fb-PMT) for 3 weeks. Efficacy wasdetermined by tumor weight and luminescent signal intensity comparedwith a control group. Further, treatment efficacy was also evaluated incomparison with known potential treatment Cilengitide. Efficacy was alsodetermined again after an additional 3 week period with no additionaltreatment.

FIG. 30 shows the effect of exemplary Compound 2 (fb-PMT) on tumorweight after 3 weeks of treatment as well as after 3 weeks of treatmentfollowed by 3 weeks off treatment. Results are shown for doses of 1mg/kg, 3 mg/kg, 6 mg/kg, and 10 mg/kg versus a control group. Tumorweight for the control group after 3 weeks was approximately 600 mg. Alltreatment groups showed a dosage dependent reduction of tumor weight tounder 100 mg.

Tumor weights were also compared after 6 weeks-3 weeks of treatmentfollowed by a 3 week period with no additional treatment. The controlgroup showed an increased tumor weight of approximately 750 mg. Alltreatment groups showed a further and additional reduction of tumorweight over the 3 weeks without treatment.

FIG. 31 shows the effect of Compound 2 (fb-PMT) on luminescent signalintensity after 3 weeks of treatment as well as after 3 weeks oftreatment followed by 3 weeks off treatment. Results are shown for dosesof 1 mg/kg, 3 mg/kg, 6 mg/kg, and 10 mg/kg versus a control group.Luminescent signal intensity for the control group after 3 weeks wasapproximately 600,000p/s. All treatment groups showed dosage dependentreduction of luminescent signal intensity to under 100,000p/s.

Signal intensity was also compared after 6 weeks-3 weeks of treatmentfollowed by a 3 week period with no additional treatment. The controlgroup showed an increased signal intensity to over 800,000p/s. Alltreatment groups showed a further and additional reduction ofluminescent signal intensity after the 3 weeks without treatment.

FIG. 32 shows the effect of Compound 2 (fb-PMT) on tumor weight after 3weeks of treatment as well as after 3 weeks of treatment followed by 3weeks off treatment. Results are shown for the 6 mg/kg dose and comparedwith both a control group and a group treated with Cilengitide at 75mg/kg. Again, tumor weight for the control group after 3 weeks wasapproximately 600 mg. Both treatment groups showed reduction of tumorweight after 3 weeks of treatment. However, the group treated withCompound 2 showed substantially increased reduction when compared withthe group treated with Cilengitide. For example, as can be seen, thegroup treated with Cilengitide showed tumor weight reduction toapproximately 300 mg, while the group treated with Compound 2 showedreduction to under 50 mg.

Tumor weights were also compared after 6 weeks-3 weeks of treatmentfollowed by a 3 week period with no additional treatment. Again, thecontrol group showed an increased tumor weight of approximately 750 mgafter the three weeks without treatment. Further, after three weekswithout treatment, the Cilengitide group showed increased tumor weightwhen compared with the 3 weeks of treatment, with a final tumor weightover 400 mg. Thus, even following 3 weeks of treatment at 75 mg/kg, thetumor was active and growing for the Cilengitide group. Conversely, theCompound 2 group showed further reduction in tumor weight even after 3weeks without treatment.

FIG. 33 shows the effect of Compound 2 on luminescent signal intensityafter 3 weeks of treatment as well as after 3 weeks of treatmentfollowed by 3 weeks off treatment. Results are shown for the 6 mg/kgdose of Compound 2 (fb-PMT) and compared with both a control group and agroup treated with Cilengitide at 75 mg/kg. Again, luminescent signalintensity for the control group after 3 weeks was approximately600,000p/s. Both treatment groups showed reduction of signal intensityafter 3 weeks of treatment. However, the group treated with Compound 2showed substantially increased reduction when compared with the grouptreated with Cilengitide. For example, as can be seen, the group treatedwith Cilengitide showed signal intensity reduction to approximately300,000p/s, while the group treated with Compound 2 showed reduction tonegligible levels.

Signal intensity was also compared after 6 weeks-3 weeks of treatmentfollowed by a 3 week period with no additional treatment. Again, thecontrol group showed an increased signal intensity to over 800,000p/s.Further, after three weeks without treatment, the Cilengitide groupshowed increased signal intensity when compared with the 3 weeks oftreatment, with a final signal intensity over 500,000p/s. Thus, evenfollowing 3 weeks of treatment at 75 mg/kg, the tumor was active andgrowing for the Cilengitide group. Conversely, the Compound 2 groupshowed even further reduction in luminescent signal activity even afterthe additional 3 weeks without treatment.

As demonstrated in this study and shown in these Figures, the describedcompounds have increased therapeutic effect against glioblastoma (GBM)when compared with both control groups and known treatmentcompounds/composition that have limited blood brain barrierpermeability. As discussed above, the increased therapeutic effect maybe attributable to a complex of factors including, active transportacross the blood brain barrier due to the thyrointegrin antagonistportion of the compound, retention within the brain and specifically inthe location of the tumor due to binding of the thyrointegrin antagonistportion of the compound to integrin αvβ3 which is present andoverexpressed in brain tumors such as GBM, and the effect of thesubstituent A on the uptake across the blood brain barrier, for example,by an increased accessibility of the transporter target in someembodiments. These features contribute to an increased initial uptakeand increased retention within the brain and at the desired treatmentlocation, resulting in increased therapeutic effect.

Still further, the compounds disclosed may have increased scalability,solubility, and yield solid products or intermediates. Synthesisscalability enables efficient and cost-effective manufacturing of thecompounds and compositions for patient use. Further, compounds andcompositions must be synthesized at sufficient levels of purity in orderto be used for treatment. The existence of a compound or composition inthe form of a solid product provides improves options for purification.This is contrast to other compounds such as P—Bi-TAT described above,which yields an oil product. The solid exemplary compounds describedherein are also readily purified by normal phase chromatography onsilica gel, which is not viable for many other PEGylated molecules,including P—Bi-TAT. For example, P—Bi-TAT requires reverse phasechromatography which is not readily scalable. Likewise, aqueoussolubility facilitates certain avenues of administration, for example,injection methods such as subcutaneous injection. Thus, these featuresare often important for realization of production of acompound/composition and also for realization of effective treatmentusing the compound/composition. The disclosed compounds may beparticularly useful as potential treatments options for glioblastoma andother conditions and may be produced in quantities sufficient fortreatment dosages.

The compounds may also be prepared as compositions comprising thedisclosed compounds. Further, the compounds and/or the compositions maybe used to treat conditions such as GBM by administering atherapeutically effective amount of the compound and/or composition to apatient in need thereof, for example, a patient suffering from thecondition.

The compositions may also be used for imaging of cancer cell/tumors. Forexample, the compositions described herein may be used to image tumorswithin the brain such as glioblastoma. Imaging may be desirable fordiagnosis and/or for treatment monitoring. Moreover, the compositionsmay be used for simultaneous treatment and imaging. For example, thecompositions may demonstrate increased retention in the targeted cancercells/tumors, allowing for enhanced treatment.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

What is claimed:
 1. A compound of formula

wherein n=5-200, A is selected from the group consisting of

a 5-membered ring heteroaryl, a fused heteroaryl, a quinolone, anindole, an amide, an ester and a phenoxy wherein R1-R7 are independentlyselected from the group consisting of: H, Me, Et, iPr, nPr, nBu, iBu,secBu, tBu, C₅-C₁₂ n-alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br,I, CN, CF₃, OCF₃, CHF₂, SO₂Me, NO₂, —O-Alkyl, —O-Aryl, —CH₂—O-Alkyl,—CH₂—O-Aryl, Esters and Amide and R8 is selected from the groupconsisting of H, Me and Et.
 2. The compound of claim 1, wherein A isselected from the group consisting of:

wherein R1-R7 are independently selected from the group consisting of:H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C₅-C₁₂ n-alkyl, cyclopentyl,cyclohexyl, phenyl, F, Cl, Br, I, CN, CF₃, OCF₃, CHF₂, OCHF₂, SO₂Me,NO₂, —O-Alkyl, —O-Aryl, —CH₂—O-Alkyl, —CH₂—O-Aryl, Esters, and Amides.3. The compound of claim 2, wherein the Alkyl is selected from the groupconsisting of: Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C₅-C₁₂ n-alkyl,cyclopentyl, and cyclohexyl.
 4. The compound of claim 2, wherein theAryl is selected from the group consisting of: phenyl and phenylsubstituted with one of alkyl, F, Cl, Br, I, CN, CF₃, OCF₃, CHF₂, OCHF₂,SO₂Me, and NO₂.
 5. The compound of claim 2, wherein the Ester isselected from the group consisting of:


6. The compound of claim 2, wherein the amide is selected from the groupconsisting of:

wherein R9 and R10 are independently selected from at least one of H,Alkyl, and Aryl.
 7. The compound of claim 1, wherein A is selected fromthe group consisting of:


8. The compound of claim 1, wherein A is selected from the groupconsisting of: a 5-membered ring heteroaryl, a fused heteroaryl, aquinoline, and an indole.
 9. The compound of claim 1, wherein A is anester selected from the group consisting of:


10. The compound of claim 1, wherein A is an amide selected from thegroup consisting of:

wherein R9 and R10 are independently selected from at least one of H,Alkyl, and Aryl.
 11. The compound of claim 1, wherein A is a phenoxy.12. The compound of claim 1, wherein the substituent A is


13. The compound of claim 12, wherein the substituent A comprises ahalogen.
 14. A compound of formula

wherein n1≥0; wherein n2 is 5-200; and wherein Z is a halogen.
 15. Acompound of formula:

wherein n1≥0; wherein n2 is 5-200; wherein R1-R4 and R9 areindependently selected from the group consisting of: H, Me, Et, iPr,nPr, nBu, iBu, secBu, tBu, C₅-C₁₂ n-alkyl, cyclopentyl, cyclohexyl,phenyl, F, Cl, Br, I, CN, CF₃, OCF₃, CHF₂, OCHF₂, SO₂Me, NO₂, —O-Alkyl,—O-Aryl, —CH₂—O-Alkyl, —CH₂—O-Aryl, Esters, and Amides; wherein R10-R13are each independently selected from the group consisting of hydrogen,iodine, and an alkane group; and wherein Y is


16. A method of treating glioblastoma, pancreatic cancer or acutemyeloid leukemia, comprising: providing a compound of formula

wherein n1≥0; wherein n2 is 5-200; and wherein Z a halogen; andadministering a therapeutically effective amount of the compound to apatient in need thereof.
 17. The method of claim 16, wherein thecondition is glioblastoma.
 18. The method of claim 16, wherein thecompound has a general formula of: